DNA replication, recombination and repair (DNA-RRR) are fundamental genetic processes carried out by large multi-protein assemblies. Separation of the double helix into single strands is a key step in each of these processes and during the time it is exposed, the single-strand DNA (ssDNA) is organized and protected by a ssDNA-binding protein. The major ssDNA-binding protein in humans is Replication protein A (RPA), a heterotrimeric complex that is an essential component of DNA-RRR assemblies. RPA not only binds and protects ssDNA, but also contacts a surprisingly large number of DNA-RRR factors. RPA's ability to simultaneously bind ssDNA and multiple proteins suggests that it helps assemble and coordinate the activities of DNA-RRR proteins on the DNA substrate. Despite a wealth of biochemical data demonstrating RPA-protein interactions, few have been mapped precisely and high resolution structural data on RPA-complexes are lacking. The proposed studies will determine the specific localization, relative strength, and interplay of RPA's multiple interactions with ssDNA and DNA-RRR proteins. Aims 1-3 involve investigations of RPA with proteins that function in the early stages of DNA replication and recombination. A combination of limited proteolysis, affinity chromatography, and mass spectrometry will be used to map the interaction domains on RPA and three partner proteins. The domains will then be sub-cloned, expressed and purified for affinity measurements and three-dimensional structure determination of the complexes. Aim 4 involves investigations of how ssDNA binding influences RPA-protein interactions and vice versa. Binding affinities in ternary complexes of RPA, ssDNA and DNA-RRR proteins will be measured, and changes in protein structure and dynamics will be characterized. These studies will generate critical information about RPA's mode of action and the manner in which replication, recombination and repair assemblies process DNA. The results will provide new insights into the fundamental links between replication, recombination and repair of DNA, and how mutations in the constituent proteins can lead to cancer and other diseases.